Selection for resistance to cacao Moniliasis assisted by molecular markers : S04P06

The basidiomycete Moniliophthora rorer, the etiological agent of Moniliasis, reaches cacao plantations (Theobroma cacao) in Central and South Americas, but its occurrence is still not registered in Brazil . The disease causes losses of up to 90 % in the production of cocoa, has difficult to control and is close to the border with Brazil, threatening the Brazilian cacao industry. As part of a larger project of preventive breeding, this study aims to identify plants that have genes involved in the control of resistance to moniliasis , using microsatellite DNA markers associated with them. The material used in this study was collected from parents (clones) and progenies from CEPEC´s breeding program. A total of 74 genotypes were used in this study, being 34 parents used in CEPEC´s breeding program and 40 trees of the cross EET-183 x ( P-7xUF-273). DNA samples from these geotypes was obtained, from leaf tissue, by the method described by Doyle & Doyle (1993). The extracted DNA was amplified by PCR , and after amplification of the SSR loci, the reaction was applied to polyacrylamide gel stained with silver nitrate as described by CRESTE et al. al. (2001) with some modifications. The clones were scored based on the size of alleles at SSR loci previously associated with resistance to moniliasis . Based on results in the loci mtcCir 26 mtcCir 46, mtcCir mtcCir 236 and 240 , it was observed that 26 of the parents used are not suitable to be used in crosses with the source of resistance UF-273, because they had the allele associated with resistance, even though they are not resistant. On the other hand , the parents Ipiranga- 1, PS-13.19, Sca-6, CCN-10, CP-188, CP-49, Salobrinho-3 and CP -111 exhibited different alleles of the source of resistance, being , therefore, suitable to be involved in further crosses with the UF -273. The individuals of the segregating population EET -183 x (P- 7x UF- 273) , were genotyped for the purpose of obtaining individuals who showed the resistance alleles to moniliasis . Out of the 40 individuals used in the study, 16 had alleles associated to resistance. (Texte intégral

Classical and "omics" approaches to control witches' broom disease of cacao [W120]

In Theobroma cacao the main biotic stresses are cause by fungi, i.e Moniliophthora perniciosa (Mp), causal agent of witches' broom disease of cacao (WBD). Breeding of Mp-resistant varieties is confronted with two major difficulties at present. First, cacao resistant sources have been identified, but most of them are Scavina 6 descendants. Second, resistance from Scavina sources has shown to be unstable. The OMICS with the classical phytopatological and breeding approaches have allowed identifying genotypes with distinction in relation to WBD resistance, thus, carrying different resistance genes. New microsatellites and SNPs markers, and new QTLs (under natural and artificial inoculations) linked to WBD disease resistance have been identified. In parallel, histopatological studies of the cacao-Mp interaction revealed multiples modes of penetration of the fungus into cacao plants as well different mechanisms of resistance. The adaptability of Mp has also been considered, and advances in the understanding the breakdown of witches´ broom resistance, in Bahia, Brazil, have been achieved. Partial results of these projects and the overall strategy will be presented. (Résumé d'auteur

Selection for cacao resistance to Moniliasis assisted by molecular markers

The basidiomycete Moniliophthora roreri, the causal agent of moniliasis, occurs in many cocoa producer countries in South and Central Americas, but not yet in Brazil. The disease can cause losses of up to 90% in cocoa production, have difficult control and, given its proximity to the border with Brazil, it threatens Brazilian cocoa production. As part of a preventive breeding program, this study aims to identify parental germplasms to be involved in crosses aiming the development of varieties resistant to moniliasis and then select individual trees carrying the QTLs of resistance, assisted by markers. In order to do that, genomic DNA was extracted from 34 clones of CEPEC´s cacao breeding program with good agronomic characteristics and others with known resistance moniliasis and with QTLs mapped. Genomic DNA was extracted according to Doyle & Doyle (1991). Subsequently the DNA was amplified by PCR using primers close to the resistance genes, submitted to the SSR gel and silver stained. It was observed that some parental clones (Sca-6, Salobrinho-3, CP-184, CP-188), even being susceptible, carry the alleles of resistance in the marker loci and so should be avoided as parent in the marker assisted selection program. (Texte intégral

Classical and "omics" approaches to control witches' broom (Moniliopthora perniciosa) disease of cacao [P0997]

In Theobroma cacao the main biotic stresses are cause by fungi, i.e Moniliophthora perniciosa (Mp), causal agent of witches' broom disease of cacao (WBD). Breeding of Mp-resistant varieties is confronted with two major difficulties at present. First, cacao resistant sources have been identified, but most of them are Scavina 6 descendants. Second, resistance from Scavina sources has shown to be unstable. The OMICS with the classical phytopatological and breeding approaches have allowed identifying genotypes with distinction in relation to WBD resistance, thus, carrying different resistance genes. New microsatellites and SNPs markers, and new QTLs (under natural and artificial inoculations) linked to WBD disease resistance have been identified. In parallel, histopatological studies of the cacao-Mp interaction revealed multiples modes of penetration of the fungus into cacao plants as well different mechanisms of resistance. The adaptability of Mp has also been considered, and advances in the understanding the breakdown of witches´ broom resistance, in Bahia, Brazil, have been achieved. Partial results of these projects and the overall strategies to control WBD will be presented. (Texte intégral

QTL evolution under natural infection using a F2 Scavina-6 x ICS1 population for witches'broom resistance in Bahia, Brazil

Studies to determine the genetic bases to witches´ broom resistance were being carried out in order to identify different genetic sources of resistance and to improve the efficiency of selection using molecular markers. Scavina-6, particularly in the cocoa producing region of the Bahia state in Brazil, was initially the main resistance source. A major QTL of resistance was detected in the linkage group 9 from a F2 Scavina 6 x ICS1 progenies (Brown et al 2005 and Faleiro et al 2006). Observations related with the number of vegetative brooms and flower cushions brooms, were carried out on143 individuals replicated three times by grafting on CEPLAC-hybrids and observed over a period between 2003 and 2008. Analyses were made with SAS, mainly to adjust phenotypic values (number of brooms ) by a regression taking into account the difference of the section of each plant (vigor was made in 2008 assessing the section of the grafted portion). QTLs analyses were carried out with MapQTL.5 software. We confirm the presence of the previous QTL located in the LG9 around the mTcCIR35 marker. However, the LOD score and the percentage of variation explained by the QTL significantly decrease over time and change its position into the confidence interval. During this new period of evaluation of the disease in the field, others QTLs of resistance were also detected. The QTLs values related to these others significant QTLs of the resistance to WBD and to a QTL involved in the vigor were also presented. This result suggests a change in the relationship between the host and the pathogen. (Résumé d'auteur

Une méthode simplifiée de traitement "en vrac" pour détecter les mauvais étiquetage dans les collections de matériel génétique du cacaoyer

Les erreurs d'étiquetage sont un problème courant dans toutes les grandes collections de matériel génétique de cacaoyer dans le monde, avec des occurrences pouvant aller jusqu'à 40% dans certaines collections, ce qui peut réduire de façon spectaculaire le gain génétique potentiel réalisé par la sélection. Les erreurs d'étiquetage peuvent se produire par mauvaise identification du même clone avec des noms différents, ou de clones différents avec le même nom. En plus de ces types d'erreurs d'étiquetage, fréquemment, dans la même rangée d'une accession particulière, on trouve différents génotypes. Les marqueurs moléculaires jouent un rôle important en aidant les conservateurs de matériel génétique et les sélectionneurs à faire disparaître les erreurs d'étiquetage dans les collections de matériel génétique. L'approche actuellement utilisée consiste à faire une caractérisation moléculaire individuelle d'un ou quelques arbres par rangée ou accession. En utilisant cette approche, les erreurs d'identification au sein de l'accession sont totalement ou partiellement ignorées. Dans cette étude, une stratégie de traitement "en vrac" a été testée pour évaluer sa capacité à détecter des erreurs d'étiquetage au sein d'une accession. La stratégie de traitement "en vrac" consiste à collecter des disques foliaires de chaque arbre dans l'accession et à les mélanger ("vrac") pour faire une extraction de l'ADN. Ensuite, des marqueurs SSR (ou autres) spécifiques à cette accession sont utilisés pour génotyper l'ADN en vrac. Dans cette étude, environ 40 disques foliaires (diamètre = 0,6 cm) de IMC67, ICS-6 et Sca-6 ont été mélangés dans différentes proportions al de 1:0:0 à 19:1:0 pour les trois clones, et les mélanges ont fait l'objet d'analyses des empreintes génétiques, en utilisant deux amorces SSR (mTcCIR-33 et mTcClR-233) sur du gel de polyacrilamide coloré à l'argent. Il a été observé que même lorsqu'une erreur sur un seul arbre (ou un seul disque foliaire) se produit avec 16 résultats corrects, dans le mélange, elle peut être détectée par notre approche en vrac. Par conséquent, les rangées de matériel génétique avec des accessions mixtes peuvent être identifiés avec une seule analyse d'ADN. Comme la plupart des collections de matériel génétique mondiales gardent moins de 10 à 15 arbres par accession, la méthode peut être praticable pour l'identification des erreurs d'étiquetage au sein d'une accession. Une autre utilisation potentielle de la méthode "en vrac" est l'élimination des erreurs d'importants essais de descendants, des populations de cartographie et des champs semenciers. (Résumé d'auteur

Study of inheritance and identification of QTLs linked to Ceratocystis wilt resistance in cacao

Ceratocystis wilt (CW) in cacao, caused by Ceratocystis cacaofunesta, is a drastic disease that results in plant death. The pathogen was recently identified in the major cacao-producing region of Brazil-Bahia. There are still many unanswered questions regarding the sources and mode of inheritance to CW. Phenotypic evaluation of CW resistance has been based on qualitative measures of the disease, therefore hindering the determination of the type of inheritance involved in this pathosystem. The identification of genetic markers tightly-linked to disease resistance loci are valuable tools for the development of resistant cultivars using marker-assisted selection (MAS). Branches of 143 six year old progenies of the F2 Sca 6 x ICS 1 population were wounded by making a 3 mm deep cut with a sterile scalpel, and inoculated with a 20 ul drop of a spore suspension of 3 x 104 CFU/ml. Fifteen days after the inoculation (DAI) stems were collected and transported to the laboratory for evaluation. Stems were sliced open vertically above and below the point of inoculation and the length of the xylem discoloration (LXD) recorded. Associations between molecular markers and resistance to CW were evaluated by a simple interval mapping (SIM) and composite interval mapping (CIM) using the software MapQTL v. 5.0. Mean parental disease scores were 12.14 ± 3.5 cm for Sca 6 and 29.57 ± 6.7 cm for ICS 1. The mean disease score for the entire population was 14.22 ± 3.5 cm. Individual progenies varied from 4.28 to 33.75 cm. The length of the LXD followed a continuous distribution. The inoculation method used allowed to quantitatively phenotype the population. QTL analysis using the SIM and CIM revealed two genomic regions located in linkage groups 3 (LXD-LG3) and 9 (LXD-LG9) associated with the expression of the CW resistance with a LODmax of 2.57 and 3.1, respectively. The QTLs explained individually from 6.9 to 8.6% of the phenotypic variation. The identification of two QTLs involved in resistance to CW offers the possibility to improve the durability of resistance in cocoa by a possible accumulation of many different resistance genes located in different chromosome regions using marker-aided selection. The marker alleles used for the introgression survey on MAS can be also used for characterization of unrelated germplasm and finding new sources of resistance.. (Résumé d'auteur

Genomic selection on cacao for disease resistance : S04T08

Under favorable conditions, diseases can cause losses up to 100% in cacao. Breeding has been the most effective strategy in reducing these losses. However, breeding is limited by the: a) long generation time; b) large tree size (9 m2/tree); c) multiple measurements per year; d) low heritability; e) requirement of the presence of the pathogen; among others. Genomic selection (GS) overcomes some of these problems. In 2011, a project on genomic selection involving Ceplac and Cirad was started, aiming to implement GS on breeding for diseases resistance and yield. In 2013, a second project is being started involving Ceplac, Cirad and Catie, aiming preventive breeding for moniliasis (a disease still absent in Brazil) resistance through GS. In the first project, a training population of 380 progenies, out of 4000 trees of the third cycle of Cepec´s breeding program, and 90 founder and pedigree clones were chosen. In the second project, a training population of 470 trees was chosen among 2500 trees established in progeny trials and evaluated for moniliasis at CATIE. Founder parents (resistant to moniliasis) of this population was also introduced in Brazil and involved in crosses for preventive breeding. Prediction equations developed in Costa Rica will be used in Brazil for selection in the absence of the pathogen. Aiming to have accurate "phenotype" measurements for the prediction equations, BLUP values from historical data have been used (around 800 thousand data points, of 15 thousand trees). The overall strategy and partial results of these projects will be presented. Work supported by Agropolis Foundation, Fapesb, Capes, Ceplac. (Texte intégral

New witches broom QTL in the Sca 6 x ICS 1 F2 population

Witches´ broom disease (WBD) of cacao, caused by the basidiomicete Moniliophthora perniciosa, is one of the most important diseases of cacao, the chocolate tree (Theobroma cacao L.). The most viable and cost-effective alternative for the control of the disease is the use of resistance through the deployment of genetic materials with high productivity. In Bahia the main source of resistance has shown vulnerability to the fungal local strains, indicating that the fungus has overcome the Sca6 resistance. It has also been reported that the fungus is changing over time. This boom and bust cycle needs to be avoided by the breeder. However, plant breeders have had very limited success in developing resistant (R) cultivars. Molecular markers linked to genes for resistance to WBD may improve selection for this trait. The objective here was to test the QTL stability under artificial inoculations. One hundred and fifty individuals of the F2 (Sca 6 x ICS 1) population were inoculated with the POPF2 inocula. POPF2 inocula were derived from brooms collected on the F2 population. A drop of 20 ?L of 2x105 basidióspores/ mL was deposited at the apical meristem of each plant. After inoculation, the plants remained for 24 h in humid chamber, with temperatures around 25 ° C and 100% RH, afterwards they were transferred to the greenhouse until the end of the experiment. Symptoms were evaluated 60 days after the inoculation day, and mapping was performed with MapQTL v. 4.0. It was observed new putative QTLs, particularly in the linkage group 1 and 4, different from the one previously reported in the linkage group 9 detected previously. These results indicate a new region in the genome involved with WBD. This might be considered in the breeding program in search for new cacao lines with durable resistance. (Texte intégral

Application of genomics tools for cacao disease resistance : S04T07

Biotic plant problems are caused by living organisms, such as fungi, bacteria, viruses, nematodes, insects, mites, and animals. Plant responses to different stresses are highly complex and involve changes at the transcriptome, cellular, and physiological levels. In Theobroma cacao the main biotic stresses are cause by fungi, causing the witches' broom (Moniliophthora perniciosa) disease of cacao, Black pod (Phytophthora spp.), frost pod (Moniliophthora roreri and, recently, Ceratocysts wilt (Ceratocystis cacaofunesta). Durable resistance is the key to hamper the advance of these diseases. The OMICS with the classical phytopatological and breeding approaches have allowed: to identify putative resistance genes; to deciphering the genomics of Thebroma cacao, to discover new microsatellite and SNP markers, and to find new QTLs linked to disease resistance. These informations are being integrating in the CEPLAC' breeding program to accelerate the search for new resistance material that carries different resistant genes. In parallel, we study these diseases at the histopatological level trying to characterizing the mechanisms of resistance underneath the hosts as well gene expression in situ. The adaptability of these plant pathogens has also been considered. Advances in the understanding of the breakdown of witches´ broom resistance have been achieved. It was shown that the fungus has a high ability to evolve towards some genotypes. Partial results of these projects and the overall strategy will be presented. Work supported by CNPq, FINEP, FAPESB,RENORBIO (Texte intégral